Biochemistry I
Lecture 16
February 20,2013
10000
Lecture 16: Enzyme Mechanism - Serine Proteases
Reading: Horton: 6.7
Nelson and Cox: Chapter 6

Transition state stabilization: Entropic

Transition state stabilization: Enthaplic
Rate Enhancement
Key Terms:
 Acyl-enzyme intermediate
 Nucleophilic agent
 Catalytic triad (Ser, His, Asp)
 Substrate specificity
Serine proteases play an important role in many
processes, e.g. digestion of dietary protein, blood
clotting cascade, and in several pathways of
differentiation and development. They are generally
produced as inactive zymogens, and auto-activate by
peptide cleavage to form the active enzyme.
Proteases active in digestion include:
Trypsin
Chymotrypsin
Elastase
Reactions Catalyzed:
Serine Proteases can hydrolyze either esters
or peptide bonds:
Ester Hydrolysis: Note that the bright
yellow color of the p-nitrophenolate ion
provides a convenient way to monitor
the rate of product formation.
Peptide Hydrolysis:
1000
100
10
1
0
DDG kJ/mol
O
O
H2O
CH3
N
N
O
H
N
H3 N
O
HO
O
p-Nitrophenolate
(bright Yellow)
p-Nitrophenyl acetate
+
O
O
O
O
O
H2O
O
O
+
H3 N
O
Catalytic Residues::
Key residues are Ser 195, His 57, and Asp 102. These three residues are called
the catalytic triad. Serine is the nucleophile, for the first part of the reaction.
Nucleophile: group that is electron rich and can form bonds with electron deficient
groups. In the case of the peptide bond (or ester) the electronegativity of the oxygen
makes the carbonyl carbon electron deficient.
+
H3 N
O
O
Asp102
O
His57
O
HN
N
Catalytic Mechanism:
O H
I. Substrate binds.
Ser195
II. Nucleophilic attack of the side chain oxygen of Ser 195 on the carbonyl
carbon of the scissile bond (bond to be cleaved) forming a tetrahedral
intermediate. Assist from His 57 (proton transfer from Ser 195).
Tetrahedral-intermediate (transition state) is stabilized by amides of
O
Ser195 and Gly 193 – enthalpic stabilization of transition state.
III: Breakage of the peptide bond with assistance from His 57 (proton transfer to the new amino
terminus). Release of the first product.
IV: Acyl-intermediate: Note that the substrate is covalently attached to the active site Serine 195
V: Nucleophilic attack of water on the acyl-enzyme intermediate with assistance of His 57 and
formation of the tetrahedral intermediate.
VI: Decomposition of acyl intermediate and release of the second product. Enzyme is in the same
form as in panel I!
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20
H
N
CH3
Biochemistry I
R1
Lecture 16
O
H
OH
N
H
R2
O
H
O
H
R1
N
N
February 20,2013
N
H
Energy
R2
O
E+S (ES)
(EP2) E+P2
Acyl-E
Reaction coordinate
I
III
II
O
O
O
O
O
O
H
N
H
N
H
O
R2
H
N
N
Ser
H
N
N
H
Ser
H O
R1
N
R1
O
R2
H
Substrate
N
H
H
O
N
H
N
O Ser
R1
N
R2
O
1st Product
H
N
H
H
N
H
N
IV
VI
V
O
O
O
O
O
H
O
H
N
H
N
H
O
N
N
N
N
H
H
H
O Ser
R1
O
H
H
O
O
N
H
Ser
R1
O
O
Ser
R1
O
2nd Product
O
H
N
H
N
H
H
H
2
N
N
N
N
Biochemistry I
Lecture 16
February 20,2013
Serine Proteases – Review
Provide names and/or roles in the catalytic
mechanism for each of the labeled items:
A:
Asp102
D O
O
His57
+
H3 N
HN
Asp
C
His
B:
N
Ser
C:
N
H
D:
B
H
N
H O
Ser195
O
A
E:
E
Kinetics & Specificity:
Substrate Specificity:
Serine proteases utilize all of the intermolecular forces that we have discussed to bind their substrates. In
addition to general recognition of the peptide by H-bonds, a particular serine protease is specific for certain
amino acids. The molecular nature of this specificity can be inferred from the structure of the active site:
Trypsin cleaves after Lys and Arg residues: Asp189 Chymotrypsin cleaves after aromatic (and large
in the active site of Trypsin interacts with the positive hydrophobic) residues: The active site of the enzyme
charge on Arg and Lys.
contains a hydrophobic pocket, formed in part by
Met192 of Chymotrypsin.
Esterases and Toxins: The enzyme that degrades
acetylcholine (a neurotransmitter associated with
muscle contrations) is a serine esterase, with the same
catalytic triad shown above. The nerve gas, sarin,
inhibits this enzyme, interfering with breathing and
other important functions .
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